THE MECHANISM OF CATALYSIS AND THE INHIBITION OF THE BACILLUS-CEREUS ZINC-DEPENDENT BETA-LACTAMASE

The plot of k(cat)/K-m against pH for the Bacillus cereus 569/H beta-l actamase class B catalysed hydrolysis of benzylpenicillin and cephalos porin indicates that there are three catalytically important groups, t wo of pK(a) 5.6 +/- 0.2 and one of pK(a) 9.5 +/- 0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues fo r catalysis. These are assigned to zinc(II)-bound water and Asp-90, bo th with a pK(a) of 5.6 +/- 0.2. A thiol, N-(2'-mercaptoethyl)-2-phenyl acetamide, is an inhibitor of the class B enzyme with a K-i of 70 mu M . The pH-dependence of K-i shows similar pH inflections to those obser ved in the catalysed hydrolysis of substrates. The pH independence of K-i between pH 6 and 9 indicates that the pK(a) of zinc(II)-bound wate r must be 5.6 and not the higher pK(a) of 9.5. The kinetic solvent iso tope effect on k(cat)/K-m is 1.3 +/- 0.5 and that on k(cat) is 1.5. Th ere is no effect on reactivity by either added zinc(II) or methanol. T he possible mechanisms of action for the class B beta-lactamase are di scussed, and it is concluded that zinc(II) acts as a Lewis acid to sta bilize the dianionic form of the tetrahedral intermediate and to provi de a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumabl y, as a general acid catalyst facilitating C-N bond fission.